1. Field of the Invention
The present invention relates generally to neurology and, more particularly, to assays, such as immunoassays, for screening for compounds that specifically alter the production of various isoforms of Axcex2.
Alzheimer""s disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in all races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms or course is currently known.
The brains of individuals with AD exhibit characteristic lesions termed senile plaques and neurofibrillary tangles. Large numbers of these lesions are generally found in patients with AD in several areas of the human brain important for memory and cognitive function. Smaller numbers of these lesions in a more restricted anatomical distribution are sometimes found in the brains of aged humans who do not have clinical AD. Senile plaques and vascular amyloid deposits (amyloid angiopathy) also characterize the brains of individuals beyond a certain age with Trisomy 21 (Down""s Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D). The principal chemical constituent of the senile plaques and vascular amyloid deposits characteristic of AD and the other disorders mentioned above is a protein designated the amyloid-xcex2 peptide (Axcex2) or sometimes xcex2AP, Axcex2P or xcex2/A4. Axcex2 was first purified and a partial amino acid sequence reported in Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120:885-890. The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829. Forms of Axcex2 having amino acids beyond number 40 were first reported by Kang et al. (1987) Nature 325:733-736.
Roher et al. (1993) Proc. Natl. Acad. Sci. USA 90:10836-840 showed that Axcex2(1-42) is the major constituent in neuritic plaques, including significant amounts of isomerized and racemized aspartyl residues as their NH2-termini. The authors also reported that Axcex2(17-42) (p3(42)) predominates in diffuse (early) plaques, whereas Axcex2(1-40) is the major constituent in the meningeal vessel deposits, comprising 60% of the total Axcex2 in those vessels. Iwatsubo et al. (1994) Neuron 13:45-53 showed that Axcex242(43)-containing senile plaques are the major species of senile plaques in sporadic AD brains. Iwatsubo et al. (1995) Annals of Neurology 37:294-299 and Lemere et al. (1996) Neurobiology of Disease 3:16-32 reported that Axcex242(43) is the major constituent of senile plaques in Down""s syndrome brains and is the initially deposited Axcex2 species in the development of AD-type neuropathological legions in these patients. In addition, Gravina et al., (1995) J. Biol. Chem. 270:7013-7016 reported both biochemical and immunocytochemical evidence that Axcex242(43) peptides were the most abundant constituents of senile plaques in AD brains and exceeded the amounts of Axcex240 peptides in such plaques.
Molecular biological and protein chemical analyses conducted during the last several years have shown that Axcex2 is a small fragment of a much larger precursor protein, referred to as the xcex2-amyloid precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans. Knowledge of the structure of the gene encoding APP has demonstrated that Axcex2 arises as a peptide fragment that is cleaved from the carboxy-terminal end of APP by as-yet-unknown enzymes (proteases). The precise biochemical mechanism by which the Axcex2 fragment is cleaved from APP and subsequently deposited as amyloid plaques in the cerebral tissue and in the walls of cerebral and meningeal blood vessels is currently unknown. Importantly, Haass et al. (Nature 359:322-325) and Seubert et al. ((1992) Nature 359:325-327) discovered that essentially all cells expressing the APP gene normally secrete an array of Axcex2 peptides, and these peptides can readily be detected and assayed in cell culture fluid (conditioned media) and human biological fluids such as plasma and cerebrospinal fluid. It has subsequently been shown that these fluids contain both the more abundant Axcex240-ending peptides and the less abundant Axcex242(43)-ending peptides (Dovey et al. (1993) Neuroreport 4:1039-1042 and Vigo-Pelfrey et al. (1993) J. Neurochem. 61:1965-68).
Several lines of evidence indicate that progressive cerebral deposition of Axcex2 plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades (for review, see Schenk (1995) J. Med. Chem. 38:4141-4154, Selkoe (1994) J. Neuropath. and Exp. Neurol. 53:438-447 and Selkoe (1991) Neuron 6:487). One of the most important lines of evidence is the discovery in 1991 that missense DNA mutations in the APP gene at amino acid 717 of the 770-amino acid isoform of APP can be found in affected members but not unaffected members of several families with a genetically determined (familial) form of AD (Goate et al. (1991) Nature 349:704-706; Chartier Harlan et al. (1991) Nature 353:844-846; and Murrell et al. (1991) Science 254:97-99). Suzuki et al. (1994) xe2x80x9cAn increased percentage of long amyloid xcex2-protein secreted by familial amyloid xcex2-protein precursor (xcex2APP717) mutants,xe2x80x9d Science 264:1336-1340 subsequently showed that, compared to normal individuals, the 717 mutation causes a higher relative production of the Axcex2(1-42) peptide. In addition, a double mutation changing lysine670-methionine671 to asparagine670-leucine671 (with reference to the 770 isoform of APP) was reported in a Swedish family with familial AD in 1992 (Mullan et al. (1992) Nature Genet 1:345-347) and is referred to as the Swedish APP variant.
Genetic linkage analyses have demonstrated that the aforementioned mutations are the specific molecular cause of AD in the members of such families that carry these mutant APP genes. In addition, a mutation at amino acid 693 of the 770-amino acid isoform of APP has been identified as the cause of the Axcex2 deposition disease, Hereditary Cerebral Hemorrhage With Amyloidosis Dutch type (HCHWA-D), and a mutation from alanine to glycine at amino acid 692 appears to cause the phenotype of AD in some family members and the phenotype of HCHWA-D in others. The discovery of these APP mutations in genetically based cases of AD argues that genetic alteration of APP and subsequent deposition of its Axcex2 fragment can cause AD.
Recently, evidence has accumulated suggesting that Axcex2(42) plays the key role in the process of senile plaque formation in AD. First, in vitro data demonstrate that Axcex2(42) accelerates the formation of Axcex2 fibrils (and thus senile plaques) by a nucleation dependent mechanism (Jarrett et al. (1993) Biochemistry 32:4693-4697). Second, while accounting for xe2x89xa610% of total Axcex2 secreted from cells (roughly 90% is Axcex2(40) (Dovey et al. (1993) Neuroreport 4:1039-1042; Asami-Odaka et al. (1995) xe2x80x9cLong amyloid xcex2-protein secreted from wild-type human neuroblastoma IMR-32 cells.xe2x80x9d Biochemistry 34:10272-10278), Axcex2(42) is the major plaque component (Kang et al. (1987) Nature 325:733-736; Iwatsubo et al. (1994) Neuron 13:45-53; Iwatsubo et al. (1995) Ann. Neurol. 37:294-299; Gravina et al. (1995) J. Biol. Chem. 270:7013-7016; Lemere et al. (1996) Neurobiology of Disease 3:16-3.2). Furthermore, all 3 early onset familial AD genes identified to date have been shown to lead to an increase in cellular secretion of Axcex2(42). Only the Swedish APP missense mutation increases the secretion of both Axcex2(40) and Axcex2(42) peptides (Dovey et al. (1993) Neuroreport 4:1039-1042, whereas the APP717 mutations and the presenilin mutations appear not to increase Axcex2(40) peptides (Suzuki et al. (1994) Science 264:1336-1340; Scheuner et al. (1995) Neurosci. Abstracts in press). Thus, the longer Axcex2(42) peptide appears to be a prime target for therapeutic intervention. However, none of the proteases involved in the major steps of APP processing have been definitively identified, including xcex3-secretase, the protease which generates the C-terminus of Axcex2. It has generally been assumed that the same protease(s) generate both Axcex2(40) and Axcex2(42) and it has been shown that both forms share a common secretory mechanism which involves acidic intracellular compartments such as the late Golgi or early endosomes (Koo and Squazzo (1994) J. Biol. Chem. 269:17386-17389; Asami-Odaka et al. (1995) Biochemistry 34:10272-10278). Recently, Higaki et al. ((1995) Neuron, 14:651-659) have shown that the Calpain inhibitor, MDL 28170, inhibits the production of both total Axcex2 and total p3 and leads to an accumulation of their respective 12 kDa and 10 kDa APP precursor fragments in treated cells. These data suggest that the compound directly inhibits at least some form of xcex3-secretase although no data are provided as to what specific form of Axcex2 and p3 are affected.
Despite the progress which has been made in understanding the underlying mechanisms of AD, there remains a need for assays to identify candidate compounds for preventing or treating the disease.
According to current theory, the processing of APP is believed to involve several specific cleavages by proteases. The enzyme that cleaves APP between amino acids 671/672 (referring to the xcex2APP770 isoform) is called xcex2-secretase. The enzyme that cleaves between amino acids 687/688 of APP (16/17 of Axcex2) is called xcex1-secretase. Until now it was believed that cleavage of APP that yielded Axcex2(40) and Axcex2(42) was carried out by a single enzyme called xcex3-secretase. However, we have discovered that a compound can inhibit the production of Axcex2(40) but not Axcex2(42). In particular, we have discovered that compounds, thought to inhibit the production Axcex2 in general, actually inhibit production of Axcex2(40) but not Axcex2(42). This indicates that multiple xcex3-secretase mechanisms are at work which can be pharmacologically dissociated.
Because Axcex2(42) is the major component of xcex2-amyloid plaques and initiates amyloid plaque formation in AD patients, it is important to have tools to screen compounds to identify those that specifically inhibit the production of Axcex2(42) and Axcex2(40), either simultaneously or separately. The current invention provides such assays.
This invention provides methods for determining whether a compound alters the amount of at least one Axcex2(x-xe2x89xa741) peptide produced by a cell. The methods involve administering the compound to a culture comprising the cell; measuring the amount of the Axcex2(x-xe2x89xa741) peptide, specifically, in a sample from the culture; and determining whether the measured amount is different than the amount expected in a sample from a culture comprising the cell to which no compound has been administered. A difference between the measured amount and the expected amount indicates that the compound alters the amount of an Axcex2(x-xe2x89xa741) peptide produced by the cell.
In another aspect, this invention provides methods for determining whether a compound alters the amount of at least one Axcex2(x-xe2x89xa741) peptide produced by a cell and alters the amount of either total Axcex2 or at least one Axcex2(x-xe2x89xa640) peptide produced by the cell. The methods involve administering the compound to a culture comprising the cell; measuring the amount of the Axcex2(x-xe2x89xa741) peptide, specifically, in a sample from the culture; measuring the amount of total Axcex2 or the Axcex2(x-xe2x89xa640) peptide, specifically, in a sample from the culture; and determining whether the measured amounts are different than the amounts expected in a sample from a culture comprising the cell to which no compound has been administered. Differences between the measured amounts and the expected amounts indicate that the compound alters the amount of the Axcex2(x-xe2x89xa741) peptide by a cell and/or the amount of total Axcex2 or the Axcex2(x-xe2x89xa640) peptide by the cell.
In one embodiment, the amount of the Axcex2 peptides are measured by immunoassay and, in particular, sandwich immunoassay comprising capture binding substances bound to a solid phase and a labeled detection binding substance.
In sandwich assays for determining the amount of at least one Axcex2(x-xe2x89xa741) peptide, the capture antibody preferably is, specific for Axcex2(x-xe2x89xa741) peptides, e.g., raised against peptide NH2-Cys-NHxe2x80x94CH2xe2x80x94(CH2)5xe2x80x94CO-GLMVGGVVIA-COOH (SEQ ID NO:4). The detection binding substance in this assay can be an antibody specific for Axcex2 peptides whose amino-terminal amino acid is no. 1 of Axcex2, or can be specific for an epitope within the junction region of Axcex2. In another embodiment the capture binding substance for measuring the amount of at least one of Axcex2(x-xe2x89xa741) peptide is specific for an epitope within the junction region of Axcex2 and the detection binding substance is an antibody specific for Axcex2(x-xe2x89xa741).
In sandwich assays for determining the amount of at least one Axcex2(x-xe2x89xa640) peptide, the capture binding substance preferably is an antibody specific for Axcex2(x-xe2x89xa640) peptides, e.g., raised against the peptide NH2-Cys-NHxe2x80x94CH2xe2x80x94(CH2)5xe2x80x94CO-GLMVGGVV-COOH (SEQ ID NO:5). The labeled detection binding substance can be an antibody specific for the Axcex2 peptides whose amino-terminal amino acid is no. 1 of Axcex2 or an antibody specific for an epitope within the junction region of Axcex2. In another embodiment the capture binding substance for measuring the amount of at least one of Axcex2(x-xe2x89xa640) peptide is specific for an epitope within the junction region of Axcex2 and the detection binding substance is an antibody specific for Axcex2(x-xe2x89xa640).
In sandwich assays for determining the amount of total Axcex2, the capture binding substance preferably is an antibody specific for an epitope within the junction region of Axcex2. The detection binding substance preferably is specific for Axcex2 peptides whose amino-terminal amino acid is no. 1 of Axcex2.
In another embodiment of an immunoassay, the step of measuring the amount of the Axcex2(x-xe2x89xa741) peptide, total Axcex2 or the Axcex2(x-xe2x89xa640) peptide in a sample from the culture comprises: pulsing the culture with a radioactive label for protein; chasing the culture without a radioactive label; administering the compound to the cell during the chase period; contacting a sample from the culture with a binding substance specific for Axcex2(x-xe2x89xa741) peptides; contacting a sample from the culture with a binding substance specific for total Axcex2 or Axcex2(x-xe2x89xa640) peptide; and determining the amount of radioactive label attached to the binding substances.
In other embodiments of the methods the culture comprises primary human neurons, primary neurons from a transgenic PDAPP mouse (i.e., a transgenic mouse whose cells harbor a PDAPP construct), a 293 human kidney cell line, a human neuroglioma cell line, a human HeLa cell line, a primary endothelial cell line, a primary human fibroblast line, a primary lymphoblast line, human mixed brain cells, or a Chinese hamster ovary (CHO) cell line. In one embodiment the cell is a host cell transfected with a recombinant expression vector encoding a human APP, e.g., a Hardy mutation such as V717F or the Swedish mutant; causing the cell to overproduce Axcex2(x-xe2x89xa741) peptides. In another aspect, the methods further comprise the step of determining whether the compound is toxic to the cell.
In another aspect this invention provides kits for specifically detecting at least one Axcex2(x-xe2x89xa741) peptide and at least one Axcex2(x-xe2x89xa640) peptide in a sample. The kits include a binding substance specific for Axcex2(x-xe2x89xa741) peptides; and a binding substance specific for Axcex2(x-xe2x89xa640) peptides.
In another aspect this invention provides kits for specifically detecting at least one Axcex2(x-xe2x89xa741) peptide and either total Axcex2 or at least one Axcex2(x-xe2x89xa640) peptide in a sample in a sandwich immunoassay. The kits include at least two different binding substances for measuring the amount of Axcex2(x-41) peptide; and at least two different binding substances for measuring the amount of total Axcex2 or Axcex2(x-xe2x89xa640) peptides.
In another aspect this invention provides methods for determining whether a compound alters the amount of at least one Axcex2(x-xe2x89xa741) peptide produced by a non-human mammal and alters the amount of either total Axcex2 or at least one Axcex2(x-xe2x89xa640) peptide produced in the non-human mammal. The methods involve measuring a first amount of the Axcex2(x-xe2x89xa741) peptide in a sample from a non-human animal used as a model of Alzheimer""s disease; measuring a first amount of total Axcex2 or the Axcex2(x-xe2x89xa640) peptide in a sample from the non-human animal; administering the compound to the non-human animal; measuring a second amount of the Axcex2(x-xe2x89xa741) peptide in a sample from the non-human animal; measuring a second amount of total Axcex2 or the Axcex2(x-xe2x89xa640) peptide in a sample from the non-human animal; and comparing the first amounts with the second amounts. The comparison indicates whether the compound increases, decreases, or leaves unchanged the amount of the Axcex2(x-xe2x89xa741) peptide and increases, decreases, or leaves unchanged the amount of the Axcex2(x-xe2x89xa640) peptide. In certain embodiments the non-human animal is a rodent, in particular, a mouse. The non-human animal can harbor a copy of an expressible transgene sequence which encodes a Hardy mutation (e.g., V717F) or the Swedish mutation of human xcex2-amyloid precursor protein (APP).